| Endonuclease | Optimal Buffer(**) | Other Buffers |
| EcoRV | None | 2xT(100%); O,G(50-100%) |
| EcoRI | Red | O(100%);R(100%)(*);2xT(100%) |
| BcuI/SpeI | Tango | B(50-100%);G(50-100%) |
| XbaI | Tango | B,G,2xT(50-100%) |
| PstI | Orange | R(100%); B,G,T,2xT(50-100%) |
| DpnI | Tango | B,G(100%): O,R,2xT(50-100%) |
(*)Star Activity
(**)Optimal Buffer from Fermentas
Use pUC19 plasmid as test, it has cut sites for EcoRI, PstI, XbaI (unsure about BcuI/SpeI, DpnI but will try anyways), and none for EcoRV
Red Buffer: EcoRI, PstI, Control (No Enzyme)
Tango Buffer: BcuI/SpeI, XbaI, DpnI, Control (No Enzyme)
Methods:
Set up Master Mixes:
| Red MM | per tube (µL) | Total (µL) |
| MilliQ H20 | 13.75 | 55 |
| Red Buffer (10x) | 2 | 7 |
| pUC19 (10pg/µL) | 2 | 7 |
| Total | 19.75 | 69 |
| Tango MM | per tube (µL) | Total (µL) |
| MilliQ H20 | 13.75 | 55 |
| Tango Buffer (10x) | 2 | 7 |
| pUC19 (10pg/µL) | 2 | 7 |
| Total | 19.75 | 69 |
To each tube, add 19.75µL of master mix and 0.25µL of enzyme
Incubated reaction mixes at 37oC (Start:7:00pm; End:7:45pm)
Add 3.3µL of 6x loading dye to each reaction mixture and load 10µL final volume onto a 1% agarose (in TAE) gel.
Add 1µL of 6x loading dye to 1µL of GeneRuler 1kb ladder (at 0.5µg/µL)
Gel loading order as follows:
| Lane | Sample |
| 1 | 1kb Ladder |
| 2 | Tango Control |
| 3 | DpnI (Tango) |
| 4 | BcuI/SpeI (Tango) |
| 5 | XbaI (Tango) |
| 6 | EcoRI (Red) |
| 7 | PstI (Red) |
| 8 | Red Control |
| 9 | Empty |
| 10 | Empty |
Ran gel at 100V for 1 hour
Results: pUC19 plasmid DNA not present at a high enough concentration to visualize by ethidium bromide staining (1kb ladder did stain).
Conclusion: Will have to re-run experiment with DNA that is present at high enough concentrations to visualize by ethidium bromide staining
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